The ability of fibroblasts to differentiate into myofibroblasts (defined by expression of alpha-SM-actin and other SM-related proteins) is well documented and thought to be essential in the pathophysiology of a variety of vascular and fibrotic diseases. Little, however, is known regarding the role of hypoxia in regulating fibroblast differentiation despite the fact that microvascular ischemia and/or global hypoxia are considered critical initiators of fibroblast activation in the systemic and pulmonary circulations, respectively. We therefore performed a series of experiments to examine the possibility that hypoxia directly induces differentiation of fibroblasts into myofibroblasts. We observed increases in the expression of SM-related proteins, especially alpha-SM-actin but also SM22alpha by adventitial fibroblasts in the pulmonary artery (PA) adventitia of chronically hypoxic animals. We found that moderate levels of hypoxia induced actin polymerization and increased expression of alpha-SM-actin, largely through Galphai initiated Rho kinase-, PI3K- and JNK-dependent signaling pathways cultured PA fibroblasts. We also found this differentiation program in fibroblasts could be induced by extracellular ATP whose concentration was increased by hypoxia. Preliminary results also demonstrated that hypoxia-induced changes in cell phenotype were attenuated by agents which inhibits extracellular ATP signaling. We also found that TGFbeta induces alpha-SM-actin expression in cultured PA adventitial fibroblasts, but that hypoxia induced alpha-SM-actin expression occurred through pathways which appear largely independent of TGFbeta. The overall goal of this proposal is thus to determine the molecular mechanisms and signaling pathways through which hypoxia induces the differentiation of fibroblasts into myofibroblasts and to determine if extracellular ATP and/or TGFbeta are important modulators of this response. In addition, since it is known that the mechanisms regulating many SM-related genes differ depending on cell type and the stimulus imposed, we will determine whether hypoxia-induced alpha-SM-actin expression in vascular adventitial fibroblast requires transcription factors that differ from those used by vascular SMC.